This Phase I SBIR Grant focuses on advancement of a new class of inhibitors of serine-based b-lactamases (Ambler classes A, C and D), for combination with the only subclass of b-lactams (the monobactams) that are not sensitive to zinc-based enzymes (Ambler Class B). Full coverage against all classes of b-lactamases is critically important in light of the alarming increase in clinical strains of Enterobacteriaceae carrying mixed serine- and metallo-enzymes. The combination product will have utility both in the general ward and ICU for treating infections with multi-drug resistant gram negative pathogens. b-Lactam antibiotics, including penicillins, cephalosporins, monobactams and carbapenems, are the most widely used antibiotic class in the U.S., accounting for more than 50% of antibacterial prescriptions. In response to pressure from 7 decades of b-lactam usage, bacteria have evolved to produce enzymes (b- lactamases) that efficiently hydrolyze and inactivate b-lactams. The past 30 years has seen the explosive growth of variants of these enzymes, with over 890 unique b-lactamases identified in naturally occurring bacterial isolates to date. Legacy inhibitors of b-lactamases (e.g., clavulanic acid and tazobactam) were designed to address enzymes that predominated at the time of their release;i.e., Class A extended spectrum b-lactamases). They do not inhibit class B, C or D enzymes, and they are inactive against Class A carbapenemases. Indeed, no relevant inhibitors of class B metallo-b-lactamases (MBLs) are in development or have been reported. The only option for preserving the b-lactam class of antibiotics in Enterobacteriaceae expressing both serine- and metallo-b-lactamases is to pair a new generation, broad-spectrum BLI that covers all serine-b-lactamases with a monobactam (e.g., Aztreonam: Azt) that is impervious to MBLs. We have identified an Advanced Lead series of boronic-acid based BLIs with coverage extending across Ambler Class A, C and D enzymes. When tested with Azt, the combination product demonstrates potent inhibition of carbapenem-resistant strains of Enterobacteriaceae that express both an MBL and at least one serine-b-lactamase. The overall goal of this Phase I SBIR grant application is to continue chemical optimization of this novel series in order to deliver Finalist compounds in advance of Preclinical Candidate selection. Such Finalists will have broad coverage of serine-b-lactamases and will rescue Azt in strains of Enterobacteriaceae that express both a Class B and a Class A, C and/or D enzyme(s). They will have high aqueous solubility (for formulating with water-soluble b-lactam antibiotics) and will rescue Azt in lethal mouse models of multi-drug resistant Enterobacteriaceae infection. Success in these endeavors will trigger the submission of a Phase II application that will drive the Finalists to Preclinical Candidate Selection and then through Preclinical Development (GMP scale-up, GLP toxicology, formulation development, epidemiology, etc), ultimately to IND submission. PUBLIC HEALTH RELEVANCE: The alarming expansion of strains of Enterobacteriaceae that co-express both metallo- and serine-b- lactamases places the future utility of the b-lactam class of antibiotics at significant risk. There is no currently- available combination b-lactam/b-lactamase inhibitor (BLI) product approved or in development that addresses this growing threat. Herein we propose to advance a new combination platform: a novel class of BLIs with activity against all Ambler classes of serine-b-lactamases (Classes A, C, and D) in combination with a monobactam antibiotic (aztreonam) that is intrinsically stable to hydrolysis by metallo-b-lactamases (Class B). Success would provide the first b-lactam/BLI product combination to cover Enterobacteriaceae expressing all types of b-lactamases. Such a product would dramatically enhance the clinician's armamentarium in the general ward and ICU against multi-drug resistant gram negative pathogens.